Role of LDL receptor protein-1 (LRP-1) in Alzheimer&#39;s disease

ABSTRACT

Brain endothelial low-density lipoprotein receptor related protein-1 (LRP-1) mediates vascular clearance of Alzheimer&#39;s amyloid-β peptide (Aβ) from the brain. Transport of Aβ occurs across the blood-brain barrier (BBB) to the systemic circulation, but the brain endothelium is compromised in Alzheimer&#39;s disease. The invention is used to diagnose the disease in symptomatic and asymptomatic individuals, to identify those at risk for disease or already affected thereby, to determine the stage of disease or its progression, to intervene earlier in or alter the disease&#39;s natural history, to provide a target for therapeutic or prophylactic treatments, to screen drugs or compare medical regimens, to determine the effectiveness of a drug or medical regimen, or any combination thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in-part of U.S. application Ser. No. 10/296,168, filed Jun. 30, 2003, pending; which was filed under 35 U.S.C. 371 as the national stage of Int'l Appln. No. PCT/US01/16561, filed May 23, 2001; which claims the benefit of provisional U.S. Appln. No. 60/206,428 filed May 23, 2000 and U.S. Appln. No. 60/246,268 filed Nov. 6, 2000.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] The U.S. government has certain rights in this invention as provided for by the terms of grants awarded by the National Institutes of Health.

FIELD OF THE INVENTION

[0003] The invention relates to the role of the vascular system in clearing Alzheimer's amyloid-β peptide (Aβ) from the central nervous system (CNS) as mediated by low-density lipoprotein receptor related protein-1 (LRP-1). Transport of Aβ occurs across the blood-brain barrier (BBB) to the systemic circulation, but the brain endothelium is compromised in Alzheimer's disease. The invention is used to diagnose symptomatic and asymptomatic individuals, to identify those at risk for disease or already affected thereby, to determine the stage of disease or its progression, to intervene earlier in or alter the disease's natural history, to provide a target for therapeutic or prophylactic treatments, to screen drugs or compare medical regimens, to determine the effectiveness of a drug or medical regimen, or any combination thereof.

BACKGROUND OF THE INVENTION

[0004] Deposition of amyloid-β peptide (Aβ) in brain occurs during normal aging and is accelerated in individuals with Alzheimer's disease (AD). Aβ is central to pathology of AD, and is the main constituent of brain parenchymal and vascular amyloid (1-6). Aβ extracted from senile plaques contains mainly Aβ₁₋₄₀ and Aβ₁₋₄₂ (7), while vascular amyloid is predominantly Aβ₁₋₃₉ and Aβ₁₋₄₀ (8). Several sequences of Aβ were found in both lesions (9-11). A major soluble form of Aβ, which is present in the blood, cerebrospinal fluid (CSF) (12-14) and brain (15-16) is Aβ₁₋₄₀. In the circulation, CSF and brain interstitial fluid (ISF), soluble Aβ may exist as a free peptide and/or associated with different transport binding proteins such as apolipoprotein J (apoJ) (17-18), apolipoprotein E (apoE) (19), transthyretin (20), lipoproteins (21), albumin (22), and alpha-2 macroglobulin (α₂M) (23).

[0005] The neuronal theory argues that soluble brain-derived Aβ is a precursor of Aβ deposits. Neuronal cells secrete Aβ in culture (24), which supports this view. An increase in soluble Aβ in AD and Down's Syndrome brains precedes amyloid plaque formation (15, 25, 26), and correlates with the development of vascular pathology (27). Several cytosolic proteases that may degrade intracellular Aβ in vitro cannot degrade extracellular Aβ from brain ISF (28) or CSF (29) in vivo. An exception is enkephalinase that may degrade Aβ₁₋₄₂ from brain ISF (28). However, the physiological importance of this degradation in vivo remains still unclear since the peptide was studied at extremely high pharmacological concentrations (30).

[0006] It has been suggested that decreased clearance of Aβ from brain and CSF is the main cause of Aβ accumulation in sporadic AD (31). Since Aβ is continuously produced in the brain, a working hypothesis in this study was that efficient clearance mechanism(s) must exist at the blood-brain barrier (BBB) to prevent its accumulation and subsequent aggregation in the brain. Cell surface receptors such as the receptor for advanced glycation end products (RAGE) (32-33), scavenger type A receptor (SR-A) (34), low-density lipoprotein receptor-related protein-1 (LRP-1) (35-38) and LRP-2 (39) bind Aβ at low nanomolar concentrations as free peptide (e.g., RAGE, SR-A), and/or in complex with (α₂M, apoE or apoJ (e.g., LRP-1, LRP-2). RAGE and SR-A regulate brain endothelial endocytosis and transcytosis of Aβ initiated at the luminal side of the BBB (33), while LRP-2 mediates BBB transport of plasma Aβ complexed to apoJ (39). Recent work shows that overexpression of functional LRP-1 minireceptors in neurons of Alzheimer's PDAPP mice results in an age-dependent increase of soluble Aβ in the brain (73) suggesting that LRP-1 on neurons in vivo does not mediate Aβ clearance from the brain. The role of vascular receptors and BBB transport in the removal of brain-derived Aβ was previously unknown.

[0007] In the present study, a technique to measure brain tissue clearance in mice was developed based on a previous model in the rabbit (40). This technique was used to determine in vivo the efflux rates of Aβ₁₋₄₀ from the CNS as a function of time and concentration of peptide, and to characterize vascular transport and/or receptor-mediated efflux mechanism(s) involved in elimination of brain-derived Aβ across the BBB. The study focused on LRP-1 and its ligands, (α₂M and apoE because both promote Aβ clearance in smooth muscle cells (35), neurons (36, 38), and fibroblasts (37); and the apoE4 genetic locus is definitely, and the α₂M genetic locus is possibly associated with increasing the risk for AD (41-42). Reduced LRP-1-mediated Aβ transport across the BBB was associated with AD. At pathological concentrations (greater than 1 μM), Aβ promotes LRP-1 degradation in brain endothelium consistent with the reduced LRP-1 brain capillary levels observed in AD. Thus, loss of LRP-1 at the BBB mediate brain accumulation of neurotoxic Aβ. This loss can be prevented.

[0008] This study can be used to improve the understanding of the pathogenesis of Alzheimer's disease and mechanisms of disease. New and nonobvious modes of diagnosis and treatment are suggested by this discovery. Other advantages of the invention are discussed below or would be apparent to persons in the art from the disclosure herein.

SUMMARY OF THE INVENTION

[0009] In one embodiment of the invention, reagents are provided in kit form that can be used for performing the methods such as the following: diagnosis, identification of those at risk for disease or already affected, or determination of the stage of disease or its progression. In addition, the reagents may be used in methods related to the treatment of disease such as the following: evaluation whether or not it is desirable to intervene in the disease's natural history, alteration of the course of disease, early intervention to halt or slow progression, promotion of recovery or maintenance of function, provision of targets for beneficial therapy or prophylaxis, comparison of candidate drugs or medical regimens, or determination of the effectiveness of a drug or medical regimen. Instructions for performing these methods, reference values and positive/negative controls, and relational databases containing patient information (e.g., genotype, medical history, symptoms, transcription or translation yields from gene expression, physiological or pathological findings) are other products that can be considered aspects of the invention.

[0010] In other embodiments of the invention, these methods for diagnosis and treatment are provided. For screening of drugs and clinical trials, the respective drug and medical regimen selected are also considered embodiments of the invention. The amount and extent of treatment administered to a cell, tissue, or individual in need of therapy or prophylaxis is effective in treating the affected cell, tissue, or individual. One or more properties/functions of affected endothelium or cells thereof, or the number/severity of symptoms of affected individuals, may be improved, reduced, normalized, ameliorated, or otherwise successfully treated. The invention may be used alone or in combination with other known methods. Instructions for performing these methods, reference values and positive/negative controls, and relational data-bases containing patient information are considered further aspects of the invention. The individual may be any animal or human. Mammals, especially humans and rodent or primate models of disease, may be treated. Thus, both veterinary and medical methods are contemplated.

[0011] Further aspects of the invention will be apparent to a person skilled in the art from the following detailed description and claims, and generalizations thereto.

DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1A shows time-disappearance curves of ¹⁴C-inulin (open circles) and ¹²⁵I-Aβ₁₋₄₀ (60 nM; TCA-precipitable ¹²⁵I-radioactivity, filled circles) from the CNS following simultaneous microinjections of tracers into the caudate nucleus in mice. Each point is mean±SD, from 3 to 7 animals. FIG. 1B shows two components of ¹²⁵I-Aβ₁₋₄₀ efflux. Vascular transport across the BBB (filled triangles) and transport by ISF bulk flow (open triangles) were computed with eqs. 3 and 4, using data from FIG. 1A. FIG. 1C shows relative contributions to Aβ₁₋₄₀ efflux by transport across the BBB (open bar), diffusion by ISF bulk flow (closed bar), and retention (shaded bar) in the brain were studied at 60 nM concentrations and calculated from fractional coefficients given in Table 1.

[0013]FIG. 2 shows time-appearance curves of ¹⁴C-inulin (open circles) and ¹²⁵I-Aβ₁₋₄₀ (60 nM; TCA-precipitable ¹²⁵I-radioactivity, filled circles) in the CSF (FIG. 2A) and plasma (FIG. 2B) following simultaneous microinjections of tracers into the caudate nucleus in mice. Values are expressed as % of injected dose (% ID); each point is mean±SD from 3 to 7 animals.

[0014]FIG. 3A shows brain TCA precipitable (open bars) and TCA non-precipitable ¹²⁵I-radioactivity (solid bars) following intracerebral microinjections of ¹²⁵I-Aβ₁₋₄₀ (60 nM) into the caudate nucleus in mice expressed as a percentage of total ¹²⁵I-radioactivity in brain; mean±SD is from 3 to 5 animals. FIG. 3B shows an HPLC elution profile of brain tissue (right panel) 60 min following intracerebral microinjection of ¹²⁵I-Aβ₁₋₄₀ (60 nM). Separation was performed for 30 mg of brain tissue on a reverse-phase HPLC column, using a 30-min linear gradient of 25% to 83% acetonitrile in 0.1% TFA, pH 2. ¹²⁵I-Aβ₁₋₄₀ eluted at 52% corresponding to the elution time of Aβ₁₋₄₀ standard. The left panel shows SDS/PAGE analysis of brain tissue supernatant at 30 min (lane a) and 60 min (lane b) following intracerebral micro-injection of ¹²⁵I-Aβ₁₋₄₀ (60 nM). The radioactivity in the brain was eluted as a single peak on HPLC with the same retention time as the Aβ₁₋₄₀ standard. Aliquots of lyophilized sample were subjected to 10% Tris-tricine SDS/PAGE, transferred to a nitrocellulose membrane, and exposed to X-ray film. FIG. 3C shows plasma TCA precipitable (open bars) and TCA nonprecipitable ¹²⁵I-radioactivity (solid bars) following intracerebral microinjections of ¹²⁵I-Aβ₁₋₄₀ (60 nM) into the caudate nucleus in mice expressed as a percentage of total ¹²⁵I-radioactivity in plasma; mean±SD is from 3 to 5 animals.

[0015]FIG. 4A shows concentration-dependent clearance of Aβ₁₋₄₀ from mouse brain. Clearance was determined 30 min after simultaneous microinjections of ¹²⁵I-Aβ₁₋₄₀ at increasing concentrations (0.05 nM to 120 nM) along with ¹⁴C-inulin into the caudate nucleus. Clearance by BBB transport (filled circles) are shown separately from clearance by ISF bulk flow (open circles). FIG. 4B shows the effects with (closed bars) or without (open bars) of anti-LRP-1 antibody R777 (60 μg/ml), RAP (0.2 μM and 5 μM), anti-α₂M antibody (20 μg/ml) and anti-LRP-2 antibody Rb6286 (60 μg/ml) on brain clearance of ¹²⁵I-Aβ1-40 at 12 nM determined 30 min after simultaneous ¹²⁵I-Aβ₁₋₄₀/¹⁴C-inulin microinjections. FIG. 4C shows the effects of anti-LRP-1 antibody R777 (60 μg/ml), anti-RAGE antibody (60 μg/ml), fucoidan (100 μg/ml) and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH, 10 mM) on brain clearance of ¹²⁵I-Aβ₁₋₄₀ at higher load of 60 nM determined 30 min after simultaneous ¹²⁵I-Aβ₁₋₄₀/¹⁴C-inulin microinjections. Mean±SD from 3 to 4 animals; *−p<0.05, ns—not significant.

[0016]FIG. 5 shows the effect of apoE genotype and age on brain clearance of ¹²⁵I-Aβ₁₋₄₀. Brain clearance of ¹²⁵I-Aβ₁₋₄₀ in 2-month old and 9-month old wild-type mice and apoE KO mice studied at a lower load of ¹²⁵I-Aβ₁₋₄₀ of 12 nM (FIG. 5A) and a higher load of 60 nM (FIG. 5B). In all studies, ¹²⁵I-Aβ₁₋₄₀ (closed bars) and ¹⁴C-inulin (open bars) were injected simultaneously and clearance determined after 30 min.

[0017] Mean±SD from 3 to 4 animals; *−p<0.05, ns—not significant in comparison to 2-month old wild-type mice.

[0018]FIG. 6 shows that LRP-1 is down regulated in human brain endothelium by excess Aβ. Western blot analyses of LRP-1 β-subunit (LRP-85) expression in brain endothelium exposed for 48 hr to different concentrations of Aβ₁₋₄₀, Aβ₁₋₄₂, or mutant Aβ (Dutch/Iowa40; DIAβ₁₋₄₀) from 1 nM to 20 μM (FIGS. 6A-6C). RAP (1.25 μM) and anti-LRP-1 β-chain-specific IgG F(ab)₂ (25 μg/ml) were incubated for 1 hr prior to the addition of Aβ peptides and then for 48 hr simultaneously with different Aβ peptides (FIG. 6D). Relative intensity of LRP-1 band in brain endothelium exposed to different Aβ peptides was determined by scanning densitometry and normalized for β-actin. Mean±SEM (n=3 to 5 independent experiments); *p<0.01; **p<0.05 for the relative expression of LRP-1 levels in the presence of a given Aβ concentration compared to in the absence Aβ.

[0019]FIG. 7 shows that high Aβ levels reduce LRP-1's half-life in human brain endothelium. Cells were labeled by [³⁵S]-methionine for 1 hr and chased for the indicated times. FIG. 7A is an autoradiograph of [³⁵S]-methionine labeled LRP-1 was immunoprecipitated with anti-LRP-1 515 kDa α-subunit-specific IgG in the presence or absence of Aβ₁₋₄₂ (20 μM). FIG. 7B is a graph of LRP-1 levels from 3 independent experiments as in FIG. 7A. *p<0.05; **p<0.01 in the presence of Aβ compared to the corresponding control value. Effects of the proteasomal inhibitor MG132 (20 μM) on LRP-1 levels in the presence or absence of Aβ₁₋₄₂ (20 μM) was determined at 8 hr (FIG. 7C). FIG. 7D is an autoradiograph of [³⁵S]-methionine labeled LRP-1 immunoprecipitated with anti-LRP-1 515 kDa α-subunit specific IgG. FIG. 7E is a graph of LRP-1 levels from 3 independent experiments as in FIG. 7C. Western blot analysis for immature 600 kDa LRP-1 (LRP-600) in the presence of Aβ₁₋₄₀, Aβ₁₋₄₂ or mutant Aβ (Dutch/Iowa40, DIAβ₁₄₀) at 20 μM within 48 hr, as demonstrated with LRP-1-specific IgG directed to the C-terminal region of LRP-1 (5A6). RAP was used at 1.2 μM. Mean±SEM (n=3) in FIGS. 7B and 7D.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0020] Elimination of amyloid-β peptide (Aβ) from the brain has been poorly understood. Following intracerebral microinjections in young mice, ¹²⁵I-Aβ₁₄₋₀ was rapidly removed from the brain (t_(1/2)≦25 min) mainly by vascular transport across the blood-brain barrier (BBB) (≧74%). The efflux transport system for Aβ₁₋₄₀ at the BBB was half-saturated (K_(0.5)) at 15.3 nM and the maximal transport capacity was reached between 70 and 100 nM. Aβ₁₋₄₀ clearance was inhibited by the receptor associated protein (44%) and antibodies against low-density lipoprotein receptor-related protein-1, LRP-1 (58%) and alpha 2-macroglobulin, α₂M (25%). Clearance was significantly reduced in young (30%) and old (46%) apolipoprotein E (apoE) knockout mice, and in old, wild-type mice (55%). There was no evidence that Aβ was metabolized in brain interstitial fluid and degraded to smaller peptide fragments and amino acids prior to its transport across the BBB into the circulation. LRP-1, although abundant in brain microvessels in young mice, was down-regulated in older animals (45%). Down-regulation of vascular LRP-1 correlated with regional Aβ accumulation in brains of Alzheimer's disease patients. The BBB removes Aβ from the brain largely by an age-dependent, LRP-1-mediated transport mechanism that is influenced by α₂M and/or apoE. This mechanism appears to be impaired in Alzheimer's disease at the level of transcript or protein abundance or receptor function (e.g., time required for receptor transcytosis or recycling, efficiency of ligand transport across the BBB).

[0021] High extracellular accumulation of soluble Aβ affects LRP-1 expression at the Aβ clearance site(s) in the brain. The interaction of Aβ and LRP-1 on brain capillary membranes regulates retention of a high β-sheet content neurotoxic Aβ₁₋₄₂ and vasculotropic mutant Aβ while clearing Aβ₁₋₄₀. At pathological concentrations, Aβ down regulates LRP-1 in brain endothelium by promoting its proteasome-dependent degradation. This down regulation may be reversed by one or more inhibitors of the ubiquitin-proteasome pathway.

[0022] Preparations of endothelial cells, isolated tissues, and in vitro cell cultures are provided from brain (e.g., microvasculature) or other organs (e.g., skin) of individuals at risk for Alzheimer's disease, affected by the disease, or not. In particular, tissues like endothelium, smooth muscle, blood vessels and capillaries of the brain, temporal and leptomeningeal arteries, or any other tissues that express LRP-1 can be examined. Blood and bone marrow cells might also be used. They can be obtained as biopsy or autopsy material; cells of interest may be isolated therefrom and then cultured. Also provided are extracts of cells; at least partially purified DNA, RNA, and protein therefrom; and methods for their isolation. These reagents can be used to establish detection limits for assays, absolute amounts of gene expression that are indicative of disease or not, ratios of gene expression that are indicative of disease or not, and the significance of differences in such values. These values for positive and/or negative controls can be measured at the time of assay, before an assay, after an assay, or any combination thereof. Values may be recorded on storage medium and manipulated with computer software; storage in a database allows retrospective or prospective study. Gene expression (e.g., detected by antibody staining) and protein activity of LRP-1 (e.g., vascular clearance of Aβ from the brain to the systemic circulation) was decreased in individuals with Alzheimer's disease.

[0023] Polynucleotides representative of genes whose expression is decreased in Alzheimer's disease may be used to identify, isolate, or detect complementary polynucleotides by binding assays. Similarly, polypeptides representative of the gene products that are decreased in Alzheimer's disease may be used to identify, isolate, or detect interacting proteins by binding assays. Optionally, bound complexes including interacting proteins may be identified, isolated, or detected indirectly though a specific binding molecule (e.g., antibody) for the gene product that is decreased in Alzheimer's disease. For the receptor-ligand system studied here, LRP-1, apoE and α₂M and are interacting proteins. Candidate compounds to treat Alzheimer's disease may interact with at least one gene, transcript, or protein which is a component of the receptor-ligand system to increase receptor activity (i.e., vascular clearance of Aβ), and be screened for their ability to provide therapy or prophylaxis. These products may be used in assays (e.g., diagnostic methods) or for treatment; conveniently, they are packaged as assay kits or in pharmaceutical form.

[0024] Assaying Polynucleotides or Polypeptides

[0025] Binding of polynucleotides or polypeptides may take place in solution or on a substrate. The assay format may or may not require separation of bound from not bound. Detectable signals may be direct or indirect, attached to any part of a bound complex, measured competitively, amplified, or any combination thereof. A blocking or washing step may be interposed to improve sensitivity and/or specificity. Attachment of a polynucleotide or polypeptide, interacting protein, or specific binding molecule to a substrate before, after, or during binding results in capture of an unattached species. See U.S. Pat. Nos. 5,143,854 and 5,412,087. Abundance may be measured at the level of protein and/or transcripts of a component of the receptor-ligand system.

[0026] Polynucleotide, polypeptide, or specific binding molecule may be attached to a substrate. The substrate may be solid or porous and it may be formed as a sheet, bead, or fiber. The substrate may be made of cotton, silk, or wool; cellulose, nitro-cellulose, uncharged nylon, or positively-charged nylon; natural rubber, butyl rubber, silicone rubber, or styrenebutadiene rubber; agarose or polyacrylamide; crystalline, amorphous, or impure silica (e.g., quartz) or silicate (e.g., glass); polyacrylonitrile, polycarbonate, polyethylene, polymethyl methacrylate, polymethylpentene, polypro-pylene, polystyrene, polysulfone, polytetrafluoroethylene, polyvinylidenefluoride, polyvinyl acetate, polyvinyl chloride, or polyvinyl pyrrolidone; or combinations thereof. Optically-transparent materials are preferred so that binding can be monitored and signal transmitted by light.

[0027] Such reagents would allow capture of a molecule in solution by specific interaction between the cognate molecules and then could immobilize the molecule on the substrate. Monitoring gene expression is facilitated by using an array.

[0028] Polynucleotide, polypeptide, or specific binding molecule may be synthesized in situ by solid-phase chemistry or photolithography to directly attach the nucleotides or amino acids to the substrate. Attachment of the polynucleotide, polypeptide, or specific binding molecule to the substrate may be through a reactive group as, for example, a carboxy, amino, or hydroxy radical; attachment may also be accomplished after contact printing, spotting with a pin, pipetting with a pen, or spraying with a nozzle directly onto a substrate. Alternatively, the polynucleotide, polypeptide, or specific binding molecule may be reversibly attached to the substrate by interaction of a specific binding pair (e.g., antibody-digoxygenin/hapten/peptide, biotin-avidin streptavidin, glutathione S transferase-glutathione, maltose binding protein-maltose, polyhistidine-nickel, protein A or G/immunoglobulin); cross-linking may be used if irreversible attachment is desired.

[0029] Changes in gene expression may be manifested in the cell by affecting transcripitional initiation, transcript stability, translation of transcript into protein product, protein stability, or a combination thereof. The abundance of transcript or polypeptide can be measured by techniques such as in vitro transcription, in vitro translation, Northern hybridization, nucleic acid hybridization, reverse transcription-polymerase chain reaction (RT-PCR), run-on transcription, Southern hybridization, cell surface protein labeling, metabolic protein labeling, antibody binding, immunoprecipitation (IP), enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent or histochemical staining, microscopy and digital image analysis, and fluorescence activated cell analysis or sorting (FACS).

[0030] A reporter or selectable marker gene whose protein product is easily assayed may be used for convenient detection. Reporter genes include, for example, alkaline phosphatase, β-galactosidase (LacZ), chloramphenicol acetyltransferase (CAT), β-glucoronidase (GUS), bacterial/insect/marine invertebrate luciferases (LUC), green and red fluorescent proteins (GFP and RFP, respectively), horseradish peroxidase (HRP), β-lactamase, and derivatives thereof (e.g., blue EBFP, cyan ECFP, yellow-green EYFP, destabilized GFP variants, stabilized GFP variants, or fusion variants sold as LIVING COLORS fluorescent proteins by Clontech). Reporter genes would use cognate substrates that are preferably assayed by a chromogen, fluorescent, or luminescent signal. Alternatively, assay product may be tagged with a heterologous epitope (e.g., FLAG, MYC, SV40 T antigen, glutathione transferase, hexahistidine, maltose binding protein) for which cognate antibodies or affinity resins are available.

[0031] A polynucleotide may be ligated to a linker oligonucleotide or conjugated to one member of a specific binding pair (e.g., antibody-digoxygenin/hapten/peptide epitope, biotin-avidin/streptavidin, glutathione transferase or GST-glutathione, maltose binding protein-maltose, polyhistidine-nickel, protein A/G-immunoglobulin).

[0032] The polynucleotide may be conjugated by ligation of a nucleotide sequence encoding the binding member. A polypeptide may be joined to one member of the specific binding pair by producing the fusion encoded such a ligated or conjugated polynucleotide or, alternatively, by direct chemical linkage to a reactive moiety on the binding member by chemical cross-linking. Such polynucleotides and polypeptides may be used as an affinity reagent to identify, to isolate, and to detect interactions that involve specific binding of a transcript or protein product of the expression vector.

[0033] Before or after affinity binding of the transcript or protein product, the member attached to the polynucleotide or polypeptide may be bound to its cognate binding member. This can produce a complex in solution or immobilized to a support.

[0034] Construction of Expression Vector

[0035] An expression vector is a recombinant polynucleotide that is in chemical form either a deoxyribonucleic acid (DNA) and/or a ribonucleic acid (RNA). The physical form of the expression vector may also vary in strandedness (e.g., single-stranded or double-stranded) and topology (e.g., linear or circular). The expression vector is preferably a double-stranded deoxyribonucleic acid (dsDNA) or is converted into a dsDNA after introduction into a cell (e.g., insertion of a retrovirus into a host genome as a provirus). The expression vector may include one or more regions from a mammalian gene expressed in the microvasculature, especially endothelial cells (e.g., ICAM-2, tie), or a virus (e.g., adenovirus, adeno-associated virus, cytomegalo-virus, herpes simplex virus, Moloney leukemia virus, mouse mammary tumor virus, Rous sarcoma virus, SV40 virus), as well as regions suitable for gene manipulation (e.g., selectable marker, linker with multiple recognition sites for restriction endo-nucleases, promoter for in vitro transcription, primer annealing sites for in vitro replication). The expression vector may be associated with proteins and other nucleic acids in a carrier (e.g., packaged in a viral particle).

[0036] The expression vector further comprises a regulatory region for gene expression (e.g., promoter, enhancer, silencer, splice donor and acceptor sites, polyadenylation signal, cellular localization sequence). Transcription can be regulated by tetracyline or dimerized macrolides. The expression vector may be further comprised of one or more splice donor and acceptor sites within an expressed region; a Kozak consensus sequence upstream of an expressed region for initiation of translation; downstream of an expressed region, multiple stop codons in the three forward reading frames to ensure termination of translation, one or more mRNA degradation signals, a termination of transcription signal, a polyadenylation signal, and a 3′ cleavage signal. For expressed regions that do not contain an intron (e.g., a coding region from a cDNA), a pair of splice donor and acceptor sites may or may not be preferred. It would be useful, however, to include a mRNA degradation signal if it is desired to express one or more of the downstream regions only under the inducing condition. An origin of replication may be included that allows replication of the expression vector integrated in the host genome or as an autonomously replicating episome. Centromere and telomere sequences can also be included for the purposes of chromosomal segregation and protecting chromosomal ends from shortening, respectively. Random or targeted integration into the host genome is more likely to ensure maintenance of the expression vector but episomes could be maintained by selective pressure or, alternatively, may be preferred for those applications in which the expression vector is present only transiently.

[0037] An expressed region may be derived from a gene encoding LRP-1 or a ligand thereof in operative linkage with a regulatory region (e.g., constituitive, regulated, or endothelial-specific promoter and an optional enhancer). The expressed region may encode a translational fusion. Open reading frames of regions encoding a polypeptide and at least one heterologous domain may be ligated in register. If a reporter or selectable marker is used as the heterologous domain, then expression of the fusion protein may be readily assayed or localized. The heterologous domain may be an affinity or epitope tag.

[0038] Screening of Candidate Compounds

[0039] Another aspect of the invention are chemical or genetic compounds, derivatives thereof, and compositions including same that are effective in the treatment of Alzheimer's disease and individuals at risk thereof. The amount that is administered to an individual in need of therapy or prophylaxis, formulation, and timing and route of delivery is effective to reduce the number or severity of symptoms, to slow or limit progression of symptoms, to inhibit expression of one or more genes that are transcribed at a higher level in Alzheimer's disease, to activate expression of one or more genes that are transcribed at a lower level in Alzheimer's disease, or any combination thereof. Determination of such amounts, formulations, and timing and route of drug delivery is within the skill of persons conducting in vitro assays, in vivo studies of animal models, and human clinical trials.

[0040] A screening method may comprise administering a candidate compound to an organism or incubating a candidate compound with a cell, and then determining whether or not gene expression is increased. The increase in activity may partially or fully compensate for a change that is associated with or may cause Alzheimer's disease. Gene expression may be increased at the level of rate of transcriptional initiation, rate of transcriptional elongation, stability of the transcript, translation of the transcript, rate of translational initiation, rate of translational elongation, stability of protein, rate of protein folding, proportion of protein in active conformation, functional efficiency of protein (e.g., activation or repression of transcription), or combinations thereof. See U.S. Pat. Nos. 5,071,773 and 5,262,300. High-throughput screening assays are possible.

[0041] The screening method may comprise incubating a candidate compound with a cell containing a reporter construct, the reporter construct comprising transcription regulatory region covalently linked in a cis configuration to a downstream gene encoding an assayable product; and measuring production of the assayable product. A candidate compound which increases production of the assayable product would be identified as an agent which activates gene expression. See U.S. Pat. Nos. 5,849,493 and 5,863,733.

[0042] The screening method may comprise measuring in vitro transcription from a reporter construct in the presence or absence of a candidate compound, the reporter construct comprising a transcription regulatory region; and determining whether transcription is altered by the presence of the candidate compound. In vitro transcription may be assayed using a cell-free extract, partially purified fractions of the cell-free extract, purified transcription factors or RNA polymerase, or combinations thereof. See U.S. Pat. Nos. 5,453,362; 5,534,410; 5,563,036; 5,637,686; 5,708,158; and 5,710,025.

[0043] Techniques for measuring transcriptional or translational activity in vivo are known in the art. For example, a nuclear run-on assay may be employed to measure transcription of a reporter gene. Translation of the reporter gene may be measured by determining the activity of the translation product. The activity of a reporter gene can be measured by determining one or more of the abundance of transcription of polynucleotide product (e.g., RT-PCR of GFP transcripts), translation of polypeptide product (e.g., immunoassay of GFP protein), and enzymatic activity of the reporter protein per se (e.g., fluorescence of GFP or energy transfer thereof).

[0044] Inhibition of the ubiquitin-proteasome pathway may be used to reverse down-regulated expression of LRP-1 (e.g., by aging, disease, a pathological concentration of Aβ) or to prevent its down regulation. Examples of proteasome inhibitors are PS-341 or derivatives thereof, lactacystin, MG132, MG262, ritonavir, and TMC-95A.

[0045] Genetic Compounds for Treatment

[0046] Gene activation may be achieved by inducing an expression vector containing a downstream region related to a gene that is down regulated (e.g., the full-length coding region or functional portions of the gene; hypermorphic mutants, homologs, orthologs, or paralogs thereof) or unrelated to the gene that acts to relieve suppression of gene activation (e.g., at least partially inhibiting expression of a negative regulator of the gene). Overexpression of transcription or translation, as well as over-expressing protein function, is a more direct approach to gene activation. Alternatively, the downstream expressed region may direct homologous recombination into a locus in the genome and thereby replace an endogenous transcriptional regulatory region of the gene with an expression cassette. In particular, gene expression of components of the receptor-ligand system transporting Aβ across the blood-brain barrier can be increased by introduction of an exogenous gene or activating an endogenous gene.

[0047] An expression vector may be introduced into a host mammalian cell or non-human mammal by a transfection or transgenesis technique using, for example, chemicals (e.g., calcium phosphate, DEAE-dextran, lipids, polymers), biolistics, electroporation, naked DNA technology, microinjection, or viral infection. The introduced expression vector may integrate into the host genome of the mammalian cell or non-human mammal. Many neutral and charged lipids, sterols, and other phospholipids to make lipid carrier vehicles are known. For example, neutral lipids are dioleoyl phosphatidylcholine (DOPC) and dioleoyl phosphatidyl ethanolamine (DOPE); an anionic lipid is dioleoyl phosphatidyl serine (DOPS); cationic lipids are dioleoyl trimethyl ammonium propane (DOTAP), dioctadecyldiamidoglycyl spermine (DOGS), dioleoyltrimethyl ammonium (DOTMA), and 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamide tetraacetate (DOSPER). Dipalmitoyl phosphatidylcholine (DPPC) can be incorporated to improve the efficacy and/or stability of delivery. FUGENE 6, LIPO-FECTAMINE, LIPOFECTIN, DMRIE-C, TRANSFECTAM, CELLFECTIN, PFX-1, PFX-2, PFX-3, PFX-4, PFX-5, PFX-6, PFX-7, PFX-8, TRANSFAST, TFX-10, TFX-20, TFX-50, and LIPOTAXI lipids are proprietary formulations. The polymer may be polyethylene glycol (PEG) or polyethylenimine (PEI); alternatively, polymeric materials can be formed into nanospheres or microspheres. Naked DNA technology delivers the expression vector in plasmid form to a cell, where the plasmid may or may not become integrated into the host genome, without using chemical transfecting agents (e.g., lipids, polymers) to condense the expression vector prior to introduction into the cell.

[0048] Thus, a mammalian cell may be transfected with an expression vector; also provided are transgenic nonhuman mammals. In the previously discussed alternative, a homologous region from a gene can be used to direct integration to a particular genetic locus in the host genome and thereby regulate expression of the gene at that locus. Polypeptide may be produced in vitro by culturing transfected cells; in vivo by transgenesis; or ex vivo by introducing the expression vector into allogeneic, autologous, histocompatible, or xenogeneic cells and then transplanting the transfected cells into a host organism. Special harvesting and culturing protocols will be needed for transfection and subsequent transplantation of host stem cells into a host mammal. Immunosuppression of the host mammal post-transplant or encapsulation of the host cells may be necessary to prevent rejection.

[0049] The expression vector may be used to replace the function of a gene that is down regulated or totally defective or supplement function of a partially defective gene. Thus, the cognate gene of the host may be neomorphic, hypomorphic, hypermorphic, or normal. Replacement or supplementation of function can be accomplished by the methods discussed above, and transfected mammalian cells or transgenic nonhuman mammals may be selected for high expression (e.g., assessing amount of transcribed or translated product, or physiological function of either product) of the downstream region.

[0050] Formulation of Compositions

[0051] Compounds of the invention or derivatives thereof may be used as a medicament or used to formulate a pharmaceutical composition with one or more of the utilities disclosed herein. They may be administered in vitro to cells in culture, in vivo to cells in the body, or ex vivo to cells outside of the individual that may later be returned to the body of the same individual or another. Such cells may be diaggregated or provided as solid tissue.

[0052] Compounds or derivatives thereof may be used to produce a medicament or other pharmaceutical compositions. Use of compositions which further comprise a pharmaceutically acceptable carrier and compositions which further comprise components useful for delivering the composition to an individual are known in the art. Addition of such carriers and other components to the composition of the invention is well within the level of skill in this art.

[0053] A reduced fat diet or drug therapy to lower lipids (e.g., statins) may be used to alter LRP-1 receptor function in a beneficial manner. Alternatively, the drug may be used to increase gene expression of a component of the receptor-ligand system (e.g., enhancing transcription or translation). Reducing the systemic concentration of Aβ with antibody depletion or filtration of antibody-Aβ complexes may favor transport across the blood-brain barrier. Vasodilation, angiogenesis, neovascularization, and osmotic shock may be used to increase blood flow and thereby increase removal of Aβ from the brain to the systemic circulation. Another method for increasing removal may be to increase permeability of the blood vessel with known drugs (e.g., bradykinin, histamine); tight junctions may be loosened or the width increased to increase permeability. Other methods for increasing transcytosis and recycling of LRP-1 may also be used (e.g., activators of cAMP signalling like theophylline). As illustrated by the latter, drugs may have one or more of the aforementioned beneficial affects. It should be noted that the modes of treatment described herein differ significantly from the mechanism described in U.S. Pat. No. 6,156,311 which identifies a role for low-density lipoprotein receptor related protein in endocytosis and degradation of Aβ. Another compound is a proteasome inhibitor (e.g., MG132).

[0054] Pharmaceutical compositions may be administered as a formulation adapted for passage through the blood-brain barrier or direct contact with the endothelium. Alternatively, pharmaceutical compositions may be added to the culture medium. In addition to the active compound, such compositions may contain pharmaceutically-acceptable carriers and other ingredients known to facilitate administration and/or enhance uptake (e.g., saline, dimethyl sulfoxide, lipid, polymer, affinity-based cell specific-targeting systems). The composition may be incorporated in a gel, sponge, or other permeable matrix (e.g., formed as pellets or a disk) and placed in proximity to the endothelium for sustained, local release. The composition may be administered in a single dose or in multiple doses which are administered at different times.

[0055] Pharmaceutical compositions may be administered by any known route. By way of example, the composition may be administered by a mucosal, pulmonary, topical, or other localized or systemic route (e.g., enteral and parenteral). The term “parenteral” includes subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intrathecal, and other injection or infusion techniques, without limitation.

[0056] Suitable choices in amounts and timing of doses, formulation, and routes of administration can be made with the goals of achieving a favorable response in the individual with Alzheimer's disease or at risk thereof (i.e., efficacy), and avoiding undue toxicity or other harm thereto (i.e., safety). Therefore, “effective” refers to such choices that involve routine manipulation of conditions to achieve a desired effect.

[0057] A bolus administered over a short time once a day is a convenient dosing schedule. Alternatively, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to twelve doses per day. Dosage levels of active ingredients in a pharmaceutical composition can also be varied so as to achieve a transient or sustained concentration of the compound or derivative thereof in an individual, especially in and around vascular endothelium of the brain, and to result in the desired therapeutic response or protection. But it is also within the skill of the art to start doses at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0058] The amount of compound administered is dependent upon factors known to a person skilled in the art such as bioactivity and bioavailability of the compound (e.g., half-life in the body, stability, and metabolism); chemical properties of the compound (e.g., molecular weight, hydrophobicity, and solubility); route and scheduling of administration; and the like. For systemic administration, passage of the compound or its metabolite through the blood-brain barrier is important. It will also be understood that the specific dose level to be achieved for any particular individual may depend on a variety of factors, including age, gender, health, medical history, weight, combination with one or more other drugs, and severity of disease.

[0059] The term “treatment” of Alzheimer's disease refers to, inter alia, reducing or alleviating one or more symptoms in an individual, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis, and/or preventing disease in an individual who is free therefrom as well as slowing or reducing progression of existing disease. For a given individual, improvement in a symptom, its worsening, regression, or progression may be determined by an objective or subjective measure. Efficacy of treatment may be measured as an improvement in morbidity or mortality (e.g., lengthening of survival curve for a selected population). Prophylactic methods (e.g., preventing or reducing the incidence of relapse) are also considered treatment. Treatment may also involve combination with other existing modes of treatment (e.g., ARICEPT or donepezil, EXELON or rivastigmine, anti-amyloid vaccine, mental exercise or stimulation). Thus, combination treatment with one or more other drugs and one or more other medical procedures may be practiced.

[0060] The amount which is administered to an individual is preferably an amount that does not induce toxic effects which outweigh the advantages which result from its administration. Further objectives are to reduce in number, diminish in severity, and/or otherwise relieve suffering from the symptoms of the disease as compared to recognized standards of care. The invention may also be effective against neurodegenerative disorders in general: for example, dementia, depression, confusion, Creutzfeldt-Jakob disease, Huntington's disease, Parkinson's disease, loss of motor coordination, multiple sclerosis, stroke, and syncope.

[0061] Production of compounds according to present regulations will be regulated for good laboratory practices (GLP) and good manufacturing practices (GMP) by governmental agencies (e.g., U.S. Food and Drug Administration). This requires accurate and complete recordkeeping, as well as monitoring of QA/QC. Oversight of patient protocols by agencies and institutional panels is also envisioned to ensure that informed consent is obtained; safety, bioactivity, appropriate dosage, and efficacy of products are studied in phases; results are statistically significant; and ethical guidelines are followed. Similar oversight of protocols using animal models, as well as the use of toxic chemicals, and compliance with regulations is required.

[0062] The following examples are merely illustrative of the invention, and are not intended to restrict or otherwise limit its practice.

EXAMPLES

[0063] Reagents

[0064] Wild-type and mutant Aβ (Dutch mutation is E22Q, Iowa mutation is D23N, and Dutch/Iowa double mutation is E22Q/D23N) peptides of 40 or 42 amino acids were synthesized, by solid-phase F-moc (9-fluorenylmethoxycarbonyl) amino acid chemistry, purified by reverse phase-HPLC and structurally characterized. Aliquots of the final products were lyophilized and stored at −20° C. until use. Radioiodination was carried out with Na[¹²⁵I] and lodobeads (Pierce), and the products were resolved by HPLC (39). Aliquots of radiolabeled Aβ₁₋₄₀ were kept at −20° C. for a maximum of four weeks prior to use. The HPLC analysis confirmed that more than 99% of radioactivity was present in the form of non-oxidized monomeric peptide.

[0065] Human recombinant RAP (EMD Biosciences, San Diego, Calif.); polyclonal goat anti-human LRP-1 N20 antibody which cross reacts with mouse LRP-1 (1:200, Santa Cruz Biotech, Santa Cruz, Calif.); monoclonal mouse antibody against the C-terminal domain of human LRP-1 β-chain which cross reacts with mouse LRP-1 (5A6, 1:350, 5 μg/ml; EMD Biosciences, San Diego, Calif.); monoclonal mouse antibody against human LRP-1 α-chain (8G1, 1:240, 5 αg/mi; EMD Biosciences, San Diego, Calif.); and monoclonal mouse antibodies P2-1 specific for human APP (1:1000, 1 mg/ml), 22C11 which recognizes mouse and human APP (1:100, 0.5 mg/ml; Chemicon International, Temecula, Calif.), and 66.1 specific for residues 1-8 of human Aβ (1:1000, 1 mg/ml) were used.

[0066] Brain Clearance Model in Mice

[0067] Male C57BL/6 wild-type mice, 8-10 weeks old and 9-10 months old, and male apoE knockout mice (apoE KO) on a C57BL/6 background (Taconic Farm, German-town, N.Y.), 8-10 weeks old and 9-10 months old, were studied. CNS clearance of radiolabeled Aβ₁₋₄₀ and the inert polar marker, inulin, was determined as described below (40, 43).

[0068] A stainless steel guide cannula was implanted stereotaxically into the right caudate-putamen of anesthetized mice (60 mg/kg i.p. sodium pentobarbital). Coordinates for the tip of the cannula were 0.9 mm anterior and 1.9 mm lateral to bregma and 2.9 mm below the surface of the brain. The guide cannula and screw were fixed to the skull with methylmethacrylate (Plastics One, Roanoke, Va.) and a stylet introduced into the guide cannula. Animals were observed for one week prior to radiotracer studies.

[0069] For radioisotope injection, animals were re-anesthetized and an injector cannula (Plastic One, Roanoke, Va.) attached with 24G-TEFLON tubing (Small Parts, Miami Lake, Fla.) to a 10 μl gas-tight microsyringe (Hamilton, Reno, Nev.) was used. The amount of injected tracers was determined accurately using a micrometer to measure linear displacement of the syringe plunger in the precalibrated microsyringe. A half microliter of tracer fluid containing ¹²⁵I-Aβ₁₋₄₀ at varying concentrations from 0.05 nM to 120 nM was injected over five minutes along with ¹⁴C-inulin. When the effects of different molecular reagents were tested, those were injected simultaneously with the radiolabeled peptides.

[0070] Time-response was studied with ¹²⁵I-Aβ₁₋₄₀ from 10 min to 300 min and dose-dependent effects determined at 30 min. The effects of different molecular reagents that may potentially inhibit ¹²⁵I-Aβ₁₋₄₀ clearance were studied at 30 min including: rabbit anti-human LRP-1 antibody designated as R777 that was affinity purified over Sepharose-LRP-1 heavy chain column as described (44) and that immunoprecipitates mouse LRP-1 as described (44) and blocks LRP-1 -mediated uptake of APP and thrombospondin in murine fibroblasts (45-46); receptor associated protein (RAP) (provided by Dr. Bu, Washington University); rabbit anti-mouse α₂M antibody designated as YNRMA2M that is specific for mouse α₂M as demonstrated by radial immunodiffusion and immunoelectrophoresis (Accurate Scientific Corp, Westbury, N.Y.); rabbit anti-rat gp330 affinity purified IgG designated as Rb6286 that cross reacts with mouse LRP-2 as reported (47) (provided by Dr. Scott Argraves, University of S.C.); rabbit anti-human anti-RAGE antibody that cross-reacts with mouse RAGE (32) (provided by D. Stern, Columbia University) and fucoidan (Sigma, St. Louis, Mo.).

[0071] Tissue Sampling and Radioactivity Analysis

[0072] Brain, blood and CSF were sampled and prepared for radioactivity analysis. Degradation of ¹²⁵I-Aβ₁₋₄₀ was initially studied by trichloroacetic acid (TCA) precipitation assay. Previous studies with ¹²⁵I-Aβ₁₋₄₀o demonstrated an excellent correlation between TCA and HPLC methods (33, 48-51). Brain, plasma and CSF samples were mixed with TCA (final concentration 10%), centrifuged at 14,000 rpm at 4° C. for 8 min to 10 min, and radioactivity in the precipitate, water and chloroform fractions determined in gamma counter (Wallac, Turku, Finland). The intactness of ¹²⁵I-Aβ₁₄₀ injected into the brain was greater than 97% by TCA analysis.

[0073] Degradation of ¹²⁵I-Aβ₁₋₄₀ in brain was further studied by the HPLC and SDS-PAGE analyses. Following intracerebral injections of ¹²⁵I-Aβ₁₋₄₀, brain tissue was homogenized in PBS containing protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 mM p-aminobenzamidine) and centrifuged at 100,000 g for 1 hr at 4° C. The supernatant was then lyophilized. The resulting material was dissolved in 0.005% TFA in water, pH 2, before injection onto a C4 column (Vydac, The Separation Group, Hesperia, Calif.). The separation was achieved with a 30-min linear gradient of 25% to 83% acetonitrile in 0.1 % TFA at a flow rate of 1 ml/min, as described (57). Under these conditions, the Aβ₁₋₄₀ standard eluted at 14.5 min. Column eluants were monitored at 214 nm. The eluted fractions were collected and counted. The intactness of ¹²⁵I-Aβ₁₋₄₀ injected into the brain by the HPLC analysis was greater than 97% confirming the results of TCA analysis.

[0074] For SDS-PAGE analysis, TCA precipitated samples were resuspended in 1% SDS, vortexed and incubated at 55° C. for 5 min, then neutralized, boiled for 3 min, homogenized and analyzed by electrophoresis in 10% Tris-tricine gels followed by fluorography. Lyophilized HPLC fractions were resuspended in sample buffer, neutralized, boiled and electrophoresed as previously reported (39).

[0075] Calculations of Clearance Rates

[0076] The analysis of radioactivity-disappearance curves from the brain was as reported (40, 43). The percentage of radioactivity remaining in the brain after microinjection was determined from eq. 1 as % Recovery in brain=100×(N_(b)/N_(i)) where, N_(b) is the radioactivity remaining in the brain at the end of the experiment and N_(i) is the radioactivity injected into the brain.

[0077] In all calculations, the d.p.m. values for ¹⁴C-inulin and the c.p.m. values for TCA-precipitable ¹²⁵I-radioactivity were used. Inulin was studied as a metabolically inert polar reference marker that is neither transported across the BBB nor retained by the brain (40); its clearance rate, i.e., k_(inulin), provides a measure of the ISF bulk flow and is calculated as N_(b(inulin))/N_(i(inulin))=exp (−k_(inulin)*t) (2).

[0078] For Aβ, there are two possible physiological pathways of elimination—direct transport across the BBB into the bloodstream and elimination by ISF bulk flow into the CSF and cervical lymphatics. It is also possible that Aβ is retained within the brain either by binding to its cell surface receptors directly as a free peptide and/or by binding to different transport proteins. Thus, according to the model, the fraction of Aβ remaining in the brain can be expressed as N_(b(Aβ))/N_(i(Aβ))=a₁+a₂*e^(−k1*t) (3), where a₁=k₂/(k₁+k₂) and a₂=k₁/(k₁+k₂), and k₁ and k₂ denote the fractional coefficients of total efflux from the brain and retention within the brain, respectively.

[0079] The fractional rate constant of Aβ efflux across the BBB from brain parenchyma can be calculated by knowing the fractional rate coefficient of total efflux of Aβ and inulin as k₃=k₁−k_((inulin)) (4), i.e., as the difference between the fractional rate constant for total efflux of Aβ and the fractional rate constant of inulin. The half-saturation concentration for the elimination of Aβ by transport across the BBB, k_(0.5,) was calculated from the equation [1−(N_(b)/N_(i))]*100=Cl_(max)/(k_(0.5)+N_(i)) (5), where Cl_(max) represents the maximal efflux capacity for the saturable component of Aβ clearance across the BBB corrected for peptide clearance by the ISF flow. Cl_(max) is expressed as a percentage of the injected dose, [1−(N_(b)/N_(i))]×100, cleared from brain by saturable BBB transport over 30 min.

[0080] The MLAB mathematical modeling system (Civilized Software, Silver Spring, Md.) was used to fit the compartmental model to the disappearance curves or percent recovery data with inverse square weighting.

[0081] Human Brain Endothelial Cells and Metabolic Labeling

[0082] Human brain endothelial cells (BEC) were isolated from rapid autopsies of neurologically normal young individuals after trauma. BECs were characterized and cultured, and then incubated with different Aβ isoforms at concentrations ranging from 1 nM to 20 μM within 48 hr. Cells were lysed and equal amounts of proteins (10 μg/mi) were separated by 10% denaturing polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membrane, and probed with 5A6 (β-chain) or 8G1 (α-chain) human anti-LRP-1-specific IgGs. The relative density of each protein was determined by scanning densitometry using β-actin as an internal control.

[0083] Human BEC (4×10⁵) were pulsed for 1 hr at 37° C. with 400 μCi of [35S]-methionine (greater than 1000 Ci/mmol; Perkin Elmer, Boston, Mass.) in methionine-free Dulbecco modified Eagle medium (GIBCO BRL, New York, N.Y.). Cells were chased at indicated times within 48 hr. Cell lysates were immunoprecipitated with anti-LRP-1 515 kDa α-chain specific IgG (8G1) on SDS-PAGE. The intensity of signal was quantified in pixels using Storm 860 Phosphoimager (Amersham Biosciences, Piscataway, N.J.).

[0084] Immunocytochemical Analysis in Mice

[0085] Expression of LRP-1 and α₂M in mouse brain was studied by immunohisto-chemical analysis. Fresh-frozen, acetone-fixed brain sections of 2-month old and 9-month old wild-type mice, as well as apoE KO mice were stained using anti-human LRP R777 antibody that cross reacts with mouse LRP-1 (44-46) (1.5 mg/ml; 1:300 dilution), and anti-mouse α₂M antibody (as described above 1:250 dilution). R777 was affinity purified over Sepharose-LRP-1 heavy chain column, as described (44). The number of positive vessels was counted in ten random fields by two independent blinded observers and expressed as percentage per mm² of section. The extent and intensity of staining in cellular elements was quantitated using the Universal Imaging System and NIH imaging systems. Microvessels were carefully excluded from the quantitation by suitably varying the magnitudes of measurement. The relative intensity of cellular staining (excluding the microvasculature) in brain sections of young mice was arbitrarily normalized to 1 for purposes of comparison. Routine control sections included deletion of primary antibody, deletion of secondary antibody body and the use of an irrelevant primary antibody.

[0086] Neuropathological Analysis in Humans

[0087] Three AD patients and three neurologically normal, age-matched controls from the Alzheimer's Disease Research Center (ADRC) of the University of Southern California were evaluated clinically and followed to autopsy. Included were three males and three females, ranging in age from 69 to 99 years.

[0088] Tissue blocks (1 cm³) were obtained postmortem (range 4 hr to 7 hr; mean 5 hr), fixed in 10% neutral buffered formalin, pH 7.3 (Sigma, St. Louis, Mo.) and embedded in paraffin or snap-frozen in liquid nitrogen-chilled isopentane. Tissues were sampled from the superior and middle frontal gyrus (Brodmann's area BA10), and cerebellar hemisphere.

[0089] Sections were stained with either hematoxylin and eosin or thioflavin S, a modified Bielschowsky silver impregnation method (Gallyas stain). Thioflavin S stained sections were viewed through a Zeiss fluorescence microscope with a narrow band, blue/violet filter at 400 to 455 nm. Examination was performed by two independent observers. Diagnosis of AD was according to a modified CERAD (Consortium to Establish a Registry for Alzheimer's Disease) protocol (52).

[0090] For immunocytochemical analysis, air-dryed 10 μm cryostat sections of frontal cortex (Brodmann's area BA10) were used. Immunocytochemistry was performed using avidin-biotin peroxidase complexes (ABC method, Vector Laboratories, Burlingame, Calif.). Antibodies used include: Aβ₁₋₄₀ (Chemicon, Temecula, Calif.), rabbit anti-human, 1:1000 (1 mg/ml); Aβ₁₋₄₂, rabbit anti-human 1:1000 (1 mg/ml); the mouse monoclonal antibody to the heavy chain of human LRP-1 designated as 8G1 which is specific for human LRP-1 and recognizes an epitope on the 515 kDa subunit (53), 1:300 (1.5 mg/ml); and CD105 (clone SMG) (Serotec, Oxford, England), mouse anti-human 1:100 (0.1 mg/ml). For single staining with CD105 and LRP, after incubation with primary antibody, sections were washed three times in PBS, pH 7.4 and treated with biotinylated anti-mouse IgG for 30 minutes. After three washes in PBS, slides were incubated with avidin-biotin-HRP complex for 30 minutes and washed three times in PBS. Binding was detected with a Vector SG peroxidase detection kit (blue-gray). For double labeling, after incubation with Aβ overnight at 4° C., sections were washed three times with PBS and treated with biotinylated anti-rabbit IgG, washed again, and binding was detected with Vector NovaRed. Following three washes in PBS, the second primary antibody (LRP or CD-105) was applied and staining performed as described for single label. Imaging was accomplished using a Zeiss Axiophot II microscope equipped with a Spot digital camera (Spot Diagnostics, Sterling Heights, Mich.).

[0091] Results

[0092]FIG. 1A illustrates brain radioactivity-disappearance curves of ¹⁴C-inulin and ¹²⁵I-Aβ₁₋₄₀ (TCA-precipitable ¹²⁵I-radioactivity) studied at a concentration of 60 nM. Clearance of inulin, a reference ECF maker that is neither transported across the BBB nor retained by the brain (40, 43), approximated a single exponential decay, as expected from previous studies. The clearance curve reflecting total efflux from brain of ¹²⁵I-Aβ₁₋₄₀ was bi-exponential and much lower than that for inulin, indicating significant biological transport of Aβ₁₋₄₀ out of the brain. The two components of Aβ₁₋₄₀ efflux, i.e., rapid elimination by vascular transport across the BBB into the blood and slow elimination through ISF flow, computed from FIG. 1A with eqs. 3 and 4 are illustrated in FIG. 1B. FIG. 1B indicates significantly higher clearance of Aβ by BBB transport than by ISF bulk flow.

[0093] The half-time, t_(1/2), for brain efflux of Aβ₁₋₄₀ and inulin calculated from FIG. 1A and eqs. 2 and 3 was 25.5±2.0 min and 239.0±12.5 min (Table 1), respectively, a 9.4-fold difference. The half-time of efflux of Aβ₁₋₄₀ across the BBB was 34.6±3.6 min, 6.9-fold faster than by ISF bulk flow. In addition to efflux, there was also a slow, time-dependent retention of Aβ₁₋₄₀ in brain parenchyma with a t_(1/2) of 164.5 min. As shown in Table 1, the rate k (min⁻¹) of clearance of Aβ₁₋₄₀ from the brain was 7.9-fold higher than that for inulin. The relative contributions of Aβ₁₋₄₀ efflux at 60 nM by transport across the BBB and by ISF bulk flow based on 5 hr measurements were 73.8% and 10.7% respectively, while 15.6% of the dose remained sequestered within the CNS (FIG. 1C). TABLE 1 Clearance rates, k, for ¹²⁵I-Aβ₁₋₄₀ and ¹⁴C-Inulin ¹²⁵I-Aβ₁₋₄₀ ¹⁴C-Inulin Parameter k (min⁻¹) t_(½) (min) k (min⁻¹) t_(½) (min) Total Efflux 0.0229 ±  25.5 ± 2.0* 0.0029 ± 0.0002 239 ± 12.5 0.0023* Transport by 0.0200 ±  34.6 ± 3.6 None None BBB 0.0023 Transport by 0.0029 ±   239 ± 12.5 0.0029 ± 0.0002 239 ± 12.5 ISF 0.0002 Retention in 0.0042 ± 164.5 ± 17.6 None None brain 0.0005

[0094] Data are mean±SD from 38 individual experiments; fractional coefficients, k, were calculated using eqs. 3 and 4; *−p<0.05 by Students' t-test.

[0095] Following CNS injection, both tracers reached the CSF, and the CSF time-appearance curves are shown in FIG. 2A. The amount of ¹²⁵I-Aβ₁₋₄₀ (TCA-precipitable ¹²⁵I-radioactivity) determined in the CSF was lower than that for inulin at each time point, possibly reflecting an active clearance of Aβ₁₋₄₀ from the CSF, as suggested previously (28). It is noteworthy that at each studied time point the ¹²⁵I-labeled material in the CSF was greater than 96% TCA-precipitable indicating no degradation of the peptide. Both tracers also appeared in plasma (FIG. 2B), and higher levels of ¹²⁵I-Aβ₁₋₄₀ TCA-precipitable radioactivity than of ¹⁴C-inulin radioactivity were consistent with active transport of Aβ₁₋₄₀ out of the CNS across the BBB. The absolute amounts of both tracers in the CSF and plasma were, however, low due to relatively rapid clearance from the CSF in comparison to slow ISF bulk flow (29), and significant systemic body clearance (48), respectively.

[0096]FIGS. 3A and 3B illustrate that ¹²⁵I-Aβ1-40 was not significantly degraded in brain ISF prior to its transport across the BBB as determined by TCA, HPLC and SDS-PAGE analysis of ¹²⁵I-radioactivity in brain. The TCA analysis suggest that only 4.2% to 9.9% of ¹²⁵I-radioactivity in brain was not TCA-precipitable at different time points within 270 min of intracerebral microinjection of ¹²⁵I-Aβ₁₋₄₀ (FIG. 3A). The HPLC analysis of brain radioactivity confirmed the TCA results by indicating that 93.7% of the peptide remains intact in brain ISF at 60 min (FIG. 3B, right). It is noteworthy that ¹²⁵I-Aβ₁₋₄₀ was greater than 97% intact at the time of injection as determined both by the HPLC and TCA analyses. The results were confirmed by SDS/PAGE analysis of lyophilized ali-quots of HPLC peaks of brain homogenates at different time points after ¹²⁵I-Aβ₁₋₄₀ injection that indicate single radioactive band at about 4 kDa (FIG. 3B, left). The identity of the radio-active components on gels as Aβ₁₋₄₀ peptide was confirmed by Western blot analysis using anti-Aβantibody and enhanced chemiluminescence as a detection system. More than 96% of ¹²⁵I-radioactivity in the CSF was TCA-precipitable at studied time points between 15 min and 270 min. In contrast, degradation products of ¹²⁵I-Aβ₁₋₄₀ were found in plasma (FIG. 3C); the amount of degraded ¹²⁵I-Aβ₁₋₄₀ corresponding to TCA nonpreci-pitable ¹²⁵I-radioactivity increased from 37.6% to 58.3% from 15 min to 120 min of intra-cerebral microinjection of intact ¹²⁵I-Aβ₁₋₄₀ (FIG. 3C). It is noteworthy that the amount of radioactivity in plasma after 120 min was relatively small and approached the limits of sensitivity of the TCA assay.

[0097] Clearance of Aβ in young mice was concentration-dependent (FIG. 4A). The efflux transport system was half-saturated (K_(0.5)) at 15.3 nM of Aβ_(1-40.) The plateau or maximal clearance capacity was reached between 70 and 100 nM, and further increases in Aβ concentration resulted in progressively greater retention of the peptide in the brain. In contrast, clearance of ¹⁴C-inulin did not change with increasing concentrations of Aβ suggesting a physiologically intact BBB (FIG. 4A).

[0098] The next set of experiments was designed to characterize the BBB transport system responsible for the transcytosis of Aβ. Brains were loaded with ¹²⁵I-Aβ₁₋₄₀ either at 12 nM (FIG. 4B) or 60 nM (FIG. 4C) and clearance determined at 30 min in the absence (open bars) or the presence (solid bars) of several drugs that may act as potential inhibitors and/or competitors of export. FIG. 4B indicates that both LRP-1 antibody (60 μg/ml) and RAP (200 nM) produced significant, 58% and 30% respectively, reductions in Aβ clearance from the brain in comparison to vehicle treated controls; a further decrease in Aβ clearance to 44% was obtained by increasing the concentration of RAP to 5 μM. A significant 25% inhibition in Aβ clearance was also obtained in the presence of anti-α₂M antibody (20 μg/ml). In contrast, anti-LRP-2 antibody (FIG. 4B) and anti-RAGE antibody (FIG. 4C) did not affect Aβ clearance. Fucoidan, a specific ligand for SR-A, produced a modest increase in the clearance, possibly by blocking the binding of Aβ to parenchymal SR-A receptors, thereby allowing more peptide to be available for clearance. At higher Aβ loads (FIG. 4C), anti-LRP-1 antibody produced a 53% decrease in clearance similar to that observed at a lower load (FIG. 4B), but Aβ recovery approached that of ¹⁴C-inulin, suggesting drainage of the peptide almost exclusively through ISF bulk flow. Clearance of ¹⁴C-inulin was not affected by any of the studied molecular reagents. BCH, a substrate that specifically blocks L-system for amino acids, does not affect clearance of Aβ across the BBB which excludes the possibility that ¹²⁵I-Aβ₁₋₄₀ is degraded to ¹²⁵I-tyrosine that is transported out of the CNS instead of ¹²⁵I-Aβ₁₋₄₀.

[0099] Next, the effect of apoE and aging was studied by determining Aβ clearance in 2-month old and 9-month old apoE KO mice and wild-type mice, using 12 nM (FIG. 5A) or 60 nM (FIG. 5B) of ¹²⁵I-Aβ₁₋₄₀. FIG. 5A shows that the clearance of Aβ was reduced by 30% in young apoE KO mice, and by about 55% and 40% in 9-month old wild-type and apoE KO mice, respectively. These results were confirmed at a higher load of Aβ, and observed decrease in clearance was 46% in 9-month old apoE KO mice (FIG. 5B).

[0100] The effect of different concentrations of Aβ on LRP-1 levels in primary human brain endothelial cells (BEC), a clearance site for Aβ in the brain in vivo, was studied. Aβ₁₋₄₀, Aβ₁₋₄₂, and/or mutant Aβ (Dutch/Iowa4o) at low concentrations (less than 10 nM) do not alter significantly LRP-1 expression within 48 hr. But at concentrations which were substantially higher than the levels of Aβ that can be transported via LRP-1 across the BBB (i.e., greater than or equal to 1 μM), all of the Aβ species progressively decreased LRP-1 levels in BEC, as demonstrated for LRP-1 85 kDa β-subunit (FIGS. 6A-6C) and 515 kDa α-subunit. RAP and an anti-LRP-1 specific antibody, but not a control non-immune IgG, block down-regulation of LRP-1 by a high level of Aβ suggesting that different Aβ species at pathological concentrations hypersaturate LRP-1 resulting in the receptor's down-regulation.

[0101] To examine whether ligand-induced down-regulation of LRP-1 by excess Aβ peptide is due to enhanced degradation of the receptor, pulse chase experiments were performed with human BEC and the half-life (t_(1/2)) of LRP-1 in the absence or presence of excess Aβ was determined. Aβ₁₋₄₂ (20 μM) decreases t_(1/2) for LRP-1 from 9.6 hr to 6.4 hr (FIGS. 7A-7B). A similar reduction in LRP-1 turnover rate was shown with Aβ₁₋₄₀ and mutant Aβ. MG132, an inhibitor of proteasome-dependent LRP-1 degradation, increased the t_(1/2) for LRP-1 in both control and Aβ-treated cells (FIGS. 7C-7D). This confirmed that LRP-1 delivery into the degradative pathway in brain endothelium is regulated by the proteasome pathway and that saturation of LRP-1 by Aβ accelerates its proteasome-dependent degradation. Control immunoblot analysis with an anti-LRP-1 C-terminal antibody that recognizes immature 600 kDa form of LRP-1 confirmed that the levels of immature LRP-1 are not affected by Aβ treatment (FIG. 7E). Thus, binding of internalized extracellular Aβ to immature LRP-1 in the ER compartment is not implicated in LRP-1 degradation by extracellular Aβ, as expected, in contrast to receptor's down-regulation by simultaneous overexpression of secreted ligands such as apoE, resulting in retention and degradation of LRP-1 in the ER.

[0102] Immunocytochemical studies with anti-LRP-1 R777 antibody staining (5 μg/ml) confirmed abundant expression of LRP-1 in brain microvessels (including capillaries, small venules and arterioles) and significant parenchymal cellular staining (including neurons) in 2-month old mice. There was a significant reduction in LRP-1 positive vessels in 9-month old mice in comparison to 2-month old mice; the number of LRP-1 positive vessels dropped from 94% in 2-month old to 52% in 9-month old mice. Quantitative analysis of LRP-1 positive parenchymal cells (excluding blood vessels) showed a trend towards reduced staining in older animals, though the difference was not statistically significant. Similarly, there was no significant difference in the number of α₂M-positive microvessels or parenchymal cells in brain between young and old mice. It is noteworthy that present staining for α₂M was not able to distinguish between circulating α₂M and α2M expressed on microvessels.

[0103] Frontal cortex of AD patients (Brodmann's area BA10) revealed moderate to marked neuritic plaques and Aβ deposits in all AD patients and parenchymal and vascular amyloid in two of the three AD patients. Controls revealed no neuritic plaques or Aβ in parenchyma and only meningeal vascular Aβ in one of the three patients. Staining with anti-LRP-1 monoclonal 8G1 (5 μg/ml) in the frontal cortex of control patients revealed moderate vascular staining in capillaries and arterioles, as well as neuronal staining. There was reduced LRP-1 staining in AD tissues, including regions with Aβ₁₋₄₀ or Aβ₁₋₄₂ positive plaques and vessels. However, the immediate subcortical white matter showed more robust vascular staining for LRP-1, and absence of staining for Aβ, both in AD and controls. Anti-CD105, which identifies vascular endothelium, revealed ample staining of capillaries and arterioles of frontal cortex in controls and moderately reduced numbers of stained vessels in AD tissues. Cerebellum revealed equivalent vascular staining with anti-LRP-1 and CD105 in AD and control sections and no anti-Aβ₁₋₄₀ or Aβ₁₋₄₂ positive staining was seen in either AD or control tissues.

[0104] The number of LRP-1-positive staining cerebral vessels dropped from about 45% in age-matched controls to about 12% in AD. They were barely detectable in patients with cerebrovascular Dutch-type β-amyloidosis. Based on in vitro results (see FIGS. 6 and 7), accumulation of Aβ adjacent to brain vasculature in patients with AD and with cerebrovascular β-amyloidosis may down regulate LRP-1 at the BBB in vivo.

[0105] Discussion

[0106] This study demonstrates the importance of vascular transport across the BBB in clearing Aβ from the brain into circulation. Moreover, this transport mechanism is shown to be mediated mainly by LRP-1 in brain microvascular endothelium, and that transport of brain-derived Aβ out of the CNS may be influenced by LRP-1 ligands, α₂M and apoE. This vascular clearance mechanism for Aβ is age-dependent, and lower clearance rates in older animals correlate with decreased vascular abundance of LRP-1.

[0107] The capability of BBB to remove Aβ was significant in younger animals. The elimination time, t_(1/2), for Aβ₁₋₄₀ at 60 nM was 25 min or 9.4-fold faster than for inulin, an ECF marker used to determine the ISF bulk flow rate (40). The major component of the CNS efflux of Aβ was transport across the BBB into the vascular system. The clearance of Aβ across the BBB was time- and concentration-dependent. At very low concentra-tions, i.e. less than 2 nM as found normally in mouse brain (54-55), Aβ₁₋₄₀ was elimi-nated from brain at a rate, on an average 3.5-fold faster than at a load of 60 nM that may compare to concentrations of Aβ₁₋₄₀ found in the brains of transgenic APP animals at 3 to 4 months (54-55). The efflux transport system was half-saturated at 15.3 nM of Aβ₁₋₄₀ and appears to be fully saturated at concentrations between 70 nM and 100 nM. Thus, this efflux transporter may be completely saturated by higher levels of Aβ as found in the brains of older transgenic APP animals (54-55), which in turn may lead to vascular accumulation of Aβ and development of prominent deposits of cerebrovascular amyloid, as recently described (56-57).

[0108] In this study, significant metabolism or degradation of Aβ₁₋₄₀ within 5 hr was not observed, in contrast to a recent report suggesting that Aβ₁₋₄₂ is degraded by enkephalinase (neprilysin) in brain within minutes (28). There may be a possibility that Aβ₁₋₄₀ and Aβ₁₋₄₂ are differentially processed in the brain. The physiological relevance of the proposed degradation mechanism for Aβ₁₋₄₂ (28), however, remains unclear since the peptide was studied at extremely high concentrations of about 240 μM that are not found even in the brains with severe β-amyloidosis (30). As shown by pharmacological studies, these high concentrations of Aβ may impair local BBB integrity (58-59), which in turn may contaminate brain ISF with blood and/or plasma that possesses Aβ-degrading activity (48), as confirmed in this study.

[0109] Consistent with the hypothesis that cytosolic peptidases have little access to Aβ peptides secreted or injected into brain ISF (28) or CSF (29), it has been recently reported that insulin-degrading enzyme (IDE) cannot not degrade Aβ in brain in vivo following intracerebral injection of radiolabeled peptide (28). This is in contrast to in vitro degradation of ¹²⁵I-Aβ₁₋₄₀ by IDE from brain and liver cytosol fractions (60). Since IDE is an intracellular protease, it is not surprising that IDE may not be able to process Aβ from brain ISF, in particular if peptide clearance is faster than its cellular uptake, as suggested here and in a previous study (28). It is noteworthy that brain endothelial cells in vitro (33) and astrocytes (61) do not catabolize Aβ, in contrast to activated microglial cells that secrete a specific metalloproteinase which degrades Aβ in vitro (61). Neuronal cells metabolize Aβ in vitro by an LRP-1-dependent mechanism that may require apoE or α₂M (38). The rate of this degradation, however, is about 50- to 100-fold slower than by transport across the BBB in vivo.

[0110] Transport of Aβ out of the CSF was not associated with significant degradation of peptide in the CSF (29). Lower CSF levels of Aβ₁₋₄₀ in comparison to inulin may suggest an active transport of Aβ from the CSF to the blood, possibly across the choroid plexus or leptomeningeal vessels, as shown previously (29). Higher levels of radiolabeled Aβ in the plasma relative to inulin confirm vascular transport of the peptide out of the CNS. Although, present results indicate that brain-derived Aβ could contribute to the pool of circulating peptide, the degradation in plasma, systemic metabolism and body clearance tend to reduce the levels of circulating peptide, as shown previously (48). Under these experimental conditions, the levels of radiolabeled Aβ in the circulation were two to three orders of magnitude lower than the brain levels, thus making re-entry of radiolabeled Aβ into the brain very unlikely since the blood-to-brain transport of Aβ normally operates down the concentration gradient (39, 62-65). In addition, the apoJ system that transports blood-borne Aβ into the brain is saturated under physiological conditions (64) that may facilitate the efflux of Aβ from brain. Previous studies have shown that circulating free Aβ is also metabolized during its transport across the BBB (48-49, 65-66), possibly by pericytes, which represent a major enzymatic barrier for the transport of several peptides and proteins across the BBB (67).

[0111] The affinity of neprilysin to its physiological substrates (e.g., enkephalins, tachykinins, atrial natriuretic peptide) and/or different synthetic peptides is in the low millimolar range (68). In contrast, the levels of Aβ in the brain are normally in low nanomolar range, and in transgenic models of brain amyloidosis they vary from 40 to 250 mmol/kg from 3 to 12 months (54). Thus, under physiological and/or pathological conditions, Aβ will likely bind to its high affinity cell surface receptors such as RAGE and/or SR-A and/or high affinity transport binding proteins, e.g., α₂M, apoE and apoJ, that all react with low nanomolar level of peptide corresponding to their K_(D) values.

[0112] In the present study, anti-LRP-1 antibodies inhibited Aβ₁₋₄₀ clearance by about 55%, both at lower (12 nM) and higher loads (60 nM) of the peptide, suggesting the involvement of LRP-1 in vascular elimination of Aβ from the brain. RAP, a chaperone protein that facilitates proper folding and subsequent trafficking of LRP-1 and LRP-2 (69), also inhibited Aβ clearance. RAP binds to multiple sites on LRP and antagonizes binding of all known LRP ligands to both LRP-1 and LRP-2 in vitro (69), as well as to LRP-2 in vivo at the blood side of the BBB (39). In the present study, RAP at higher concentrations produced comparable inhibition of Aβ clearance as an anti-LRP-1 antibody. It is interesting that anti-LRP-1 antibody almost completely inhibited vascular transport of Aβ at higher concentrations of peptide, which may indicate that LRP-1 could be of primary importance in eliminating the peptide from the brain. At a lower load of the peptide (i.e., 12 nM) though, neither of the molecular reagents was able to abolish clearance of Aβ, which suggests that in addition to LRP-1, there may be an alternative, highly sensitive BBB transport mechanism(s) that eliminates the peptide from the brain at very low concentrations. The molecular nature of this putative “second” transport system is not presently known, although present data suggest that RAGE and LRP-2 are unlikely to be involved in rapid elimination of Aβ from brain. The fact that fucoidan, an SR-A ligand moderately increased clearance of Aβ suggests that inhibition of SR-A receptors in brain may decrease CNS sequestration of the peptide, thus allowing more peptide to be available for enhanced clearance across the BBB.

[0113] The role of LRP-1 in promoting Aβ clearance in vitro in smooth muscle cells, neurons and fibroblasts by α₂M and apoE has been suggested (35-38), although at significantly slower rates than across the BBB, as demonstrated in the present study. High affinity in vitro binding of Aβ to α₂M and lipidated apoE3 and apoE4 and a lower affinity binding to delipidated apoE isoforms has been well documented (23, 70). Binding/uptake studies in mouse embryonic fibroblasts, wild type and deficient in LRP-1, confirmed that free Aβ is not a ligand for LRP-1 (37, 45). The possible role of the two LRP-1 ligands in elimination of Aβ by vascular transport is suggested by inhibition of Aβ clearance with anti-α₂M antibodies, and significantly reduced clearance in apoE KO animals by 30% and 46% at 2 months and 9 months of age, respectively, in comparison to wild-type young controls. In relation to these findings, it is interesting to note that recent studies indicated that lack of endogenous mouse apoE in both the APP^(V717F) and APPsw mouse models of AD results in less Aβ deposition and no fibrillar Aβ deposits in the brain (57, 71). This suggests that mouse apoE strongly facilitates Aβ fibrillogenesis. It is possible that mouse apoE also plays a role in clearance of soluble Aβ across the BBB as suggested by the current studies but that its ability to influence Aβ aggregation in APP transgenic mice is dominant. In 10 contrast to the effects of mouse apoE, a recent study demonstrates that human apoE isoforms suppresses early Aβ deposition in APP^(V717F) mice (72). Further studies in this model will be useful to determine whether this suppressive effect of human apoE isoforms on early Aβ deposition is secondary to effects on facilitating of Aβ transport across the BBB. Although the present study does not rule out the possibility that Aβ clearance by neurons, vascular smooth muscle cells and fibroblasts shown in vitro (35-38) may also occur in vivo, vascular transport across the BBB seems to be of primary importance for rapid elimination of Aβ from brain in vivo.

[0114] Since normal aging is associated with Aβ accumulation in brain (1), and there is a significant, time-dependent and progressive accumulation of Aβ with age in transgenic APP animals (54-55), it was hypothesized that the clearance mechanism of Aβ is impaired in older animals, and is also possibly impaired in elderly humans.

[0115] The present findings of about 55-65% inhibition of Aβ clearance in 9-month old wild-type animals in comparison to young (2-month old) animals confirmed this hypothesis. Immunocytochemical studies indicated a significant reduction in the number of LRP-1 positive cerebral blood vessels from 94% in 2-month old to 52% in 9-month old mice, which correlated well with the observed reductions in the clearance capacities between the two age groups. Interestingly, downregulation of vascular LRP-1 correlated well with regional parenchymal and vascular accumulation of Aβ in brains of Alzheimer's patients compared to age-matched controls. In brain areas where LRP-1 vascular expression remains prominent, as in the white matter, no accumulation of Aβ was found in Alzheimer's brains.

[0116] Moreover, excess Aβ amplifies its own accumulation in brain ISF by accelerating LRP-1's degradation at the BBB clearance site. LRP-1 on endothelium can be reduced by a high level of Aβ. This concentration-dependent effect can be prevented by RAP or anti-LRP-1 specific antibody. Cells affected or unaffected by disease, aged or young cells, cells incubated with wild-type or mutant Aβ (or aggregates thereof), normal or reduced levels of LRP-1 on cells, or combinations thereof may be used in screening assays. The effect of candidate drugs and/or medical regimens for treating Alzheimer's disease may be evaluated using such cells. For example, LRP-1 degradation, transport, and/or synthesis by be manipulated to prevent or reverse the down regulation seen in Alzheimer's disease.

[0117] These results are illustrative of the discovery that the vascular system plays an important role in regulating the levels of Aβ in the brain. The findings further suggest that if the levels of Aβ in brain extracellular space exceed the transport capacity of the clearance mechanism across the BBB, or if the vascular transport of the peptide were impaired, as for example, by down-regulation of LRP-1, this would result in accumulation of Aβ in the brain and possibly formation of amyloid plaques. Previous studies have demonstrated a major role of the BBB in determining the concentrations of Aβ in the CNS by regulating transport of circulating Aβ (33, 39, 49-51, 62-66). The present study extends this hypothesis by showing that vascular transport across the BBB out of the brain may represent a major physiological mechanism that prevents accumulation of Aβ and amyloid deposition in brain.

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[0190] All references (e.g., articles, books, patents, and patent applications) cited above are indicative of the level of skill in the art and are incorporated by reference.

[0191] All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. More-over, “comprising” allows the inclusion of other elements in the claim, “comprising essentially of” allows the inclusion of other elements in the claim that do not materially affect operation of the invention, and no particular relationship between or among elements of a claim is meant unless such limitation is explicitly recited (e.g., arrangement of components in a product claim, order of steps in a method claim).

[0192] From the foregoing, it would be apparent to a person of skill in this art that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification. 

What is claimed is:
 1. A method of at least diagnosing an individual with Alzheimer's disease or identifying an individual as being at risk for developing Alzheimer's disease, said method comprising: (a) measuring at least abundance of low-density lipoprotein receptor related protein-1 (LRP-1), abundance of transcripts thereof, or LRP-1 receptor activity in an individual, (b) comparing the abundance of LRP-1 protein, abundance of transcripts thereof, or activity of LRP-1 receptor in said individual to at least one control, and (c) at least diagnosing said individual with Alzheimer's disease or identifying said individual as being at risk for developing Alzheimer's disease when the abundance of LRP-1 protein, abundance of transcripts thereof, or activity of LRP-1 receptor in said individual is decreased relative to said control.
 2. The method of claim 1, wherein the abundance of LRP-1 protein, abundance of transcripts thereof, or activity of LRP-1 receptor is measured at least in a brain vascular or systemic endothelial cell of said individual.
 3. The method of claim 1, wherein the abundance of LRP-1 protein, abundance of transcripts thereof, or activity of LRP-1 receptor is measured at least in a brain capillary, a temporal artery, a leptomeningeal artery, or at the blood-brain barrier of said individual.
 4. The method of claim 1, wherein said at least one control is a value indicative of increased risk for developing Alzheimer's disease and/or a value indicative of decreased risk for developing Alzheimer's disease.
 5. A method of at least treating an individual with Alzheimer's disease or at risk for developing Alzheimer's disease, said method comprising increasing low-density lipoprotein receptor related protein-1 (LRP-1) mediated removal of amyloid-β peptide (Aβ) at the blood-brain barrier in said individual in the direction from brain to blood.
 6. The method of claim 5, wherein removal of Aβ is increased by increasing at least LRP-1 expression or receptor activity.
 7. The method of claim 5, wherein removal of Aβ is increased by increasing at least expression of a ligand of LRP-1 or transport of said ligand.
 8. The method of claim 7, wherein said ligand of LRP-1 is apolipoprotein E.
 9. The method of claim 7, wherein said ligand of LRP-1 is α₂-macroglobulin.
 10. The method of claim 7, wherein LRP-1 mediated removal of Aβ is increased by administering at least one proteasome inhibitor.
 11. A method of at least treating an individual with Alzheimer's disease or at risk for developing Alzheimer's disease, said method comprising increasing vascular clearance of amyloid-β peptide (Aβ) from the brain of said individual.
 12. The method of claim 11, wherein vascular permeability to Aβ is increased.
 13. The method of claim 11, wherein at least angiogenesis or neovasularization in the brain is increased.
 14. A method of determining the effectiveness of at least a drug or medical regimen for treating Alzheimer's disease, said method comprising: (a) measuring the abundance of low-density lipoprotein receptor related protein-1 (LRP-1), abundance of transcripts thereof, or activity of LRP-1 receptor in the presence of said drug or medical regimen, and (b) at least determining said drug or medical regimen is effective when the abundance of LRP-1 protein, abundance of transcripts thereof, or activity of LRP-1 receptor is increased in the presence of said drug or medical regimen.
 15. The method of claim 14 further comprising decreasing said abundance of LRP-1, abundance of transcripts thereof, or activity of LRP-1 receptor prior to treatment.
 16. The method of claim 15, wherein said abundance of LRP-1, abundance of transcripts thereof, or activity of LRP-1 receptor is decreased by at least one soluble amyloid-β peptide (Aβ) and/or aggregate thereof.
 17. A novel drug or use of a medical regimen for treating Alzheimer's disease determined to be effective by the method of claim
 14. 18. The drug or use of a medical regimen of claim 17 which comprises a proteasome inhibitor.
 19. A kit comprised of one or more of a specific binding molecule for low-density lipoprotein receptor related protein-1 (LRP-1) or a transcript thereof used to measure abundance of said LRP-1 protein, abundance of transcripts thereof, or LRP-1 receptor activity, and/or a non-Alzheimer's disease control containing LRP-1 protein or a transcript thereof. 